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, 48, 130-142

Knockdown of Vimentin Reduces Mesenchymal Phenotype of Cholangiocytes in the Mdr2 -/- Mouse Model of Primary Sclerosing Cholangitis (PSC)

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Knockdown of Vimentin Reduces Mesenchymal Phenotype of Cholangiocytes in the Mdr2 -/- Mouse Model of Primary Sclerosing Cholangitis (PSC)

Tianhao Zhou et al. EBioMedicine.

Abstract

Background: Cholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2-/- model of PSC.

Methods: In vivo studies were performed in 12 wk. Mdr2-/- male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT).

Findings: There was increased mesenchymal phenotype of cholangiocytes in Mdr2-/- mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-β1 secretion in Mdr2-/- mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-β1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels.

Interpretation: Our study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC. FUND: National Institutes of Health (NIH) awards, VA Merit awards.

Keywords: Ductular reaction; Fibroblast; Fibrosis; Senescence; Transforming growth factor beta 1.

Figures

Fig. 1
Fig. 1
Evaluation of epithelial and mesenchymal phenotypes in liver sections, isolated cholangiocytes. [a] Immunohistochemistry for vimentin in liver sections, original magn. 20×, Scale bar = 100 μm. [b] Immunofluorescence for vimentin (in green) co-stained with CK-19 (in red). Nuclei are stained with DAPI. original magn. 40×, Scale bar = 20 μm. [c] The mRNA expression of EMT markers were evaluated by qPCR in isolated cholangiocytes. *p < .05 versus WT mice; #p < .05 versus Mdr2−/− mice. [d] Immunofluorescence for E-cadherin (in green) co-stained with CK-19 (in red). Nuclei are stained with DAPI. original magn. 40×, Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Knockdown of vimentin ameliorates liver damage, ductular reaction and intrahepatic bile duct mass (IBDM). [a] Liver histology was evaluated in liver sections (4 μm) stained with hematoxylin and eosin (H&E). Observations were processed in a blinded fashion by a board-certified pathologist. Original magn. 20×, Scale bar = 100 μm. [b] Immunohistochemistry for CK-19 in liver sections, original magn. 20×, Scale bar = 100 μm. Percentage of IBDM. *p < .05 versus WT mice; #p < .05 versus Mdr2−/− mice.
Fig. 3
Fig. 3
Vimentin Morpholino treatment decreases liver fibrosis. [a] Measurement of collagen deposition by Sirius Red staining in liver sections. Orig. magn. x20, Scale bar = 100 μm. [b] Immunofluorescence for Collagen 1 (in Magenta) co-stained with CK-19 (in red) and desmin (in green). original magn. 40×, Scale bar = 20 μm. [c] The mRNA expression of fibrotic markers was evaluated by qPCR in isolated cholangiocytes. *p < .05 versus WT mice; #p < .05 versus Mdr2−/− mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Knockdown of vimentin reduces biliary senescence. [a] Measurement of cellular senescence by SA-β-gal staining in liver sections, original magn. 20×, Scale bar = 100 μm. Percentage of SA-β-gal-positive area. #p < .05 versus Mdr2−/− mice. [b] Immunofluorescence for p16 (in green) co-stained with CK-19 (in red). Nuclei are stained with DAPI. original magn. 40×, Scale bar = 20 μm. [c–d] The mRNA expression of senescent markers was evaluated by qPCR in isolated cholangiocytes and hepatic stellate cells. *p < .05 versus WT mice; #p < .05 versus Mdr2−/− mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Expression of EMT markers in human PSC patients and isolated PSC patient-derived cholangiocytes. [a] The mRNA expression of EMT markers was evaluated by qPCR in healthy controls and PSC patients. *p < .05 versus healthy controls. [b] Immunohistochemistry for vimentin (in brown) co-stained with CK-19 (in red) in human FFPE sections, original magn. 20×, Scale bar = 100 μm. [c] Western blot analyses for vimentin and other EMT markers in healthy controls and PSC patients. Protein expression levels were normalized to GAPDH. *p < .05 versus healthy controls. [d] Immunofluorescence for vimentin, CK-19 and HNF4α (in green) for hPSCL and HIBEpiC. Nuclei are stained with DAPI. original magn. 20×, Scale bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Loss of vimentin reduces mesenchymal phenotypes of cholangiocytes induced by TGF-β1 in vitro. [a–b] The mRNA expression of EMT markers (vimentin and E-cadherin), fibrotic markers (Col1a1 and Fn1) and senescent markers (p16 and p21) was evaluated by qPCR in basal HIBEpiC and control or vimentin CRISPR/Cas9 KO plasmid transfected HIBEpiC with/without TGF-β1 stimulation. *p < .05 versus basal HIBEpiC; #p < .05 versus HIBEpiC treated with TGF-β1.
Fig. 7
Fig. 7
HHSteCs treated with cholangiocyte supernatant lacking vimentin have decreased fibrotic reaction in vitro. The mRNA expression of fibrotic markers (Col1a1, Fn1 and TGF-β1), senescent markers (p16 and p21) and E-cadherin was evaluated by qPCR in HHSteCs treated with cholangiocyte supernatant collected from WT, Mdr2−/−, Mdr2−/− vimentin Vivo-Morpholino, and Mdr2−/− mismatched mice. *p < .05 vs. basal HHSteCs; #p < .05 vs. HHSteCs treated with cholangiocyte supernatant from Mdr2−/− mice.

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